We previously demonstrated that E. coli maltose binding protein (MBP) has a remarkable ability to enhance the solubility and promote the proper folding of its fusion partners. For this reason, and because MBP fusion proteins routinely accumulate to very high levels in E. coli, we have made MBP the cornerstone of our approach for high-throughput protein expression and purification. However, MBP fusion proteins do not always bind efficiently to amylose resin, and even when they do the fusion proteins are rarely pure after amylose affinity chromatography. Therefore, to compensate for the relatively poor performance of MBP as an affinity tag, we attempted to incorporate supplementary tags within the general framework of an MBP fusion protein. We identified several locations within the framework of an MBP fusion protein where accessory tags could be added without compromising the ability of MBP to promote the solubility of its fusion partners. We then designed and successfully tested a generic protocol for protein production in E. coli that utilizes a dual His6-MBP affinity tag. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. During FY2009, we have been testing this method for the production of recombinant proteins (both secreted and intracellular formats) in the yeast Kluyveromyces lactis. We have found that the yield of intracellular HisMBP fusion proteins is extremely poor in general, and secreted HisMBP fusion proteins are frequently truncated after the MBP domain. Future experiments will explore the impact of alternative fusion partners on the yield of recombinant proteins in K. lactis. Promising results have recently been obtained (2010) using green fluorescent protein as a fusion parter for both intracellular and extracellular (secretion) protein expression in K. lactis. Experiments to develop and test tools for the production of recombinant proteins in the protozoan Leishmania tarantolae are just getting underway, as is an effort to evaluate the host Brevibacillus choshinensis for the secretion of recombinant proteins. Because most affinity tags have the potential to interfere with structural studies, reliable ways to remove them are absolutely necessary. Accordingly, we have invested a substantial effort in trying to exploit the highly specific tobacco etch virus (TEV) protease for this purpose. To improve the solubility of TEV protease in E. coli, we designed an expression vector that produces the enzyme in the form of an MBP fusion protein that cleaves itself in vivo to generate an N-terminally His-tagged TEV protease catalytic domain that is free of MBP. A dramatic increase in the yield of TEV protease was realized by using a tRNA accessory plasmid to compensate for the presence of arginine codons that are rarely used in E. coli. We also devised a simple method for intracellular processing of fusion proteins by TEV protease, which is used to determine whether or not a passenger protein is likely to be properly folded when it is fused to MBP. We have shown that many different amino acid side chains can be accommodated in the P1'site of a TEV protease recognition site with little or no impact on the efficiency of processing. Consequently, in many cases it is possible to use TEV protease to produce recombinant proteins with no non-native residues attached to their N-termini. Wild-type TEV protease cleaves itself at a specific site to generate a truncated polypeptide with greatly reduced enzymatic activity. We managed to overcome the autolysis problem by constructing a mutant enzyme (S219V) that is nearly impervious to autoinactivation and almost twice as catalytically active as the wild-type enzyme. We have distributed S219V TEV protease expression vectors to hundreds of research laboratories around the world. We have also determined crystal structures of TEV protease complexed with a peptide substrate and an inhibitor, which revealed the structural basis of its stringent sequence specificity. We are currently focusing on the characterization of other highly specific proteases, such as that encoded by the tobacco vein mottling virus (TVMV), which we have recently crystallized in complex with a peptide substrate. The co-crystal structure suggested that TVMV protease should have more stringent sequence specificity in the S1'pocket, and we have been able to confirm this experimentally. More recently, we characterized a trio of alphavirus proteases encoded by Sindbis Virus, Semliki Forest Virus and Venezuelan Equine Encephalitis Virus that we hoped would be useful alternatives to TEV protease. Although these proteases were shown to have adequate specificity for use as reagents to remove affinity tags, their catalytic efficiency is far less than that of the potyviral TEV and TVMV proteases. Finally, we have been investigating the utility of a recombinant form of a fungal carboxypeptidase (MeCPA) for removing short affinity tags (e.g., polyhistidine) from the C-termini of recombinant proteins. We have carried out a thorough analysis of the enzyme's substrate specificity and shown that it is capable of being used to remove C-terminal His-tags on a preparative level for crystallography. However, the yield of MeCPA obtained from the baculovirus expression system is rather low (250 micrograms per liter), and so efforts are underway to find a more efficient way to produce the recombinant enzyme. Alternative enzymes, such as bovine carboxypeptidases A and B are also being expressed in a variety of systems. We recently (2010) developed a method for the production of MeCPA in E. coli that produces twice the yield that can be obtained from the baculovirus system, and it seems likely that substantially greater yields will can be achieved with minor modifications of the current procedure. The key was to express the enzyme as an MBP fusion protein in E. coli cells that carry mutations that transform the cytosol into a more oxidative environment, while simultaneously overproducing the protein disulfide isomerase DsbC, which normally resides in the periplasm, in the cytosol.